Corum:Whole Plasmid PCR: Difference between revisions

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==Overview==
==Overview==
Whole plasmid PCR protocol using PFU Ultra DNA polymerase.
Whole plasmid polymerase chain reaction (WP-PCR) protocol using [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase]. It is imperative to use 5'-phosphorylated primers if the nicked DNA is going to be repaired downstream with ligase. PCR should be limited to templates of about 5 kb, but the protocol could probably be pushed up to 10 kb. See the PfuUltra II fusion manual. Primers should be sense-antisense pairs. Optimal amplification occurs with 30-35 b primers at 0.5 μM final concentration. At higher concentrations, the primers bind to each other, inhibiting amplification. For longer primers, the annealing temperature will need to be adjusted from the one mentioned in this protocol.


==Materials==
==Materials==


For a 50 μL whole plasmid PCR PCR reaction:
For a 50 μL WP-PCR reaction:


* 36 μL H<sub>2</sub>O
* 37 μL H<sub>2</sub>O
* 5 μL 10X PFU Ultra PCR buffer
* 5 μL 10X PFU Ultra PCR buffer
* 5 μL 2mM (each) dNTP mix
* 5 μL 10 μM sense/antisense primer mix (0.5 μM final, each)
* 1 μL 5nM plasmid template (0.1 nM final)
* 1 μL 12.5 mM (each) dNTP mix (0.25 mM final)
* 1 μL 10μM sense primer (200nM final)
* 1 μL 5 nM plasmid template (0.1 nM final)
* 1 μL 10μM antisense primer (200nM final)
* 1 μL [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase]
* 1 μL PFU Ultra DNA polyermerase


==Procedure==
==Procedure==
# In a PCR tube, mix the components on ice in the order they are listed above.
# In a PCR tube, add the components on ice in the order they are listed above. Mix gently and spin.
# Perform the following thermocycling program:
# Perform the following thermocycling program:
## 95 °C 1 min
## Initial melting: 95 °C 2 min
## 95 °C 30 s
## Melting: 95 °C 20 s
## T<sub>H</sub> = 55 °C 30 s
## Annealing: T<sub>a</sub> °C 20 s, where T<sub>a</sub> = T<sub>m</sub> - 5 °C
## 72 °C 2 min for each 1 kb PCR product
## Elongation: 72 °C 2 min / kb template
## Repeat steps 2-4 a total of 12-36 times (24 is standard).
## Repeat steps 2-4 a total of 30 times
## 72 °C 20 min
## Final elongation: 72 °C 30 min
## 12 °C hold
## 12-16 °C hold
# Verify product with [http://openwetware.org/wiki/Corum:Gel_Electrophoresis gel electrophoresis].
# Quantify product with [http://openwetware.org/wiki/Corum:DNA_Quantification quantifluore DNA quantification].


==Notes==
==Notes==
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==Contact==
==Contact==
* [http://openwetware.org/wiki/User:Sean_P_Corum Sean P Corum]
* [http://openwetware.org/wiki/User:Sean_P_Corum Sean P Corum]
*'''SC 18:46, 23 July 2012 (EDT)''':
*'''SC 12:50, 13 December 2012 (EST)''':


or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  

Latest revision as of 11:10, 13 December 2012

Overview

Whole plasmid polymerase chain reaction (WP-PCR) protocol using PfuUltra II fusion HS DNA polymerase. It is imperative to use 5'-phosphorylated primers if the nicked DNA is going to be repaired downstream with ligase. PCR should be limited to templates of about 5 kb, but the protocol could probably be pushed up to 10 kb. See the PfuUltra II fusion manual. Primers should be sense-antisense pairs. Optimal amplification occurs with 30-35 b primers at 0.5 μM final concentration. At higher concentrations, the primers bind to each other, inhibiting amplification. For longer primers, the annealing temperature will need to be adjusted from the one mentioned in this protocol.

Materials

For a 50 μL WP-PCR reaction:

  • 37 μL H2O
  • 5 μL 10X PFU Ultra PCR buffer
  • 5 μL 10 μM sense/antisense primer mix (0.5 μM final, each)
  • 1 μL 12.5 mM (each) dNTP mix (0.25 mM final)
  • 1 μL 5 nM plasmid template (0.1 nM final)
  • 1 μL PfuUltra II fusion HS DNA polymerase

Procedure

  1. In a PCR tube, add the components on ice in the order they are listed above. Mix gently and spin.
  2. Perform the following thermocycling program:
    1. Initial melting: 95 °C 2 min
    2. Melting: 95 °C 20 s
    3. Annealing: Ta °C 20 s, where Ta = Tm - 5 °C
    4. Elongation: 72 °C 2 min / kb template
    5. Repeat steps 2-4 a total of 30 times
    6. Final elongation: 72 °C 30 min
    7. 12-16 °C hold
  3. Verify product with gel electrophoresis.
  4. Quantify product with quantifluore DNA quantification.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Relevant papers and books

  1. Sambrook, J and Russell, DW (2001) Molecular Cloning: A Laboratory Manual (Volume II) - Cold Spring Harbor Laboratory Press ISBN 0879695773

Contact

or instead, discuss this protocol.

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